Calcitonin products and therapies for treating inflammatory or degenerative diseases

ABSTRACT

Calcitonin products and therapies for treating inflammatory or degenerative diseases are disclosed herein. The pharmaceutical compositions disclosed herein include a first therapeutic agent that is calcitonin, in free or salt form; a second therapeutic agent selected from the group consisting of a protease inhibitor, an antibiotic, a non-steroidal anti-inflammatory agent, a COX-2 inhibitor and a steroidal anti-inflammatory agent other than glucocorticoid; and a pharmaceutically acceptable excipient, carrier or diluent. The methods disclosed herein for treating inflammatory or degenerative diseases in a subject include administering a therapeutically effective amount of calcitonin, in free or salt form, to the subject; and co-administering, as part of a combination therapy, a therapeutically effective amount of a second therapeutic agent selected from the group consisting of a protease inhibitor, an antibiotic, a non-steroidal anti-inflammatory agent, a COX-2 inhibitor and a steroidal anti-inflammatory agent other than glucocorticoid to the subject.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/046,501, filed Oct. 4, 2013 (now pending), which is a continuation ofU.S. patent application Ser. No. 13/238,801, filed on Sep. 21, 2011 (nowabandoned), which claims the benefit of and priority to U.S. ProvisionalPatent Application No. 61/385,078, filed on Sep. 21, 2010 (now expired),which are hereby incorporated herein by reference in their entiretiesfor the teachings therein.

FIELD

The present disclosure relates to combination therapy for the treatmentof inflammatory or degenerative diseases, and particularly to the use ofcalcitonin in combination with at least one other active agent fortreating such inflammatory or degenerative diseases, and to therapeuticproducts that include both calcitonin and at least one other activeagent.

BACKGROUND

Inflammatory or degenerative diseases, including diseases of the joints,e.g. osteoarthritis (OA), rheumatoid arthritis (RA) or juvenilerheumatoid arthritis (JRA), and including inflammation that results fromautoimmune response, e.g. lupus, ankylosing spondylitis (AS) or multiplesclerosis (MS), can lead to substantial loss of mobility due to pain andjoint destruction. Cartilage that covers and cushions bone within jointsmay become degraded over time thus undesirably permitting direct contactof two bones that can limit motion of one bone relative to the otherand/or cause damage to one by the other during motion of the joint.Subchondral bone just beneath the cartilage may also degrade. However,compounds helpful in preventing bone loss will not necessarily preventcartilage degradation. Cartilage may be degraded by proteases present insynovial fluid that covers the cartilage surface or from white bloodcells that infiltrate the joint space.

Diseases such as osteoarthritis are multimodal in nature, rendering itdifficult for any single therapeutic agent to effectively treat all ofthe symptoms of the disease. Inflammatory or degenerative diseases ofthe joints have been treated with a variety of therapeutic agents,including a range of non-steroidal anti-inflammatory drugs (NSAIDs) thatpossess both analgesic and anti-inflammatory properties, but withoutideal clinical success in many instances. The NSAIDs in particularprovide symptomatic relief but fail to adequately protect the jointsfrom further disease progression. Monotherapy to date has failed toadequately provide both a chondroprotective effect while also providingimproved mobility and reduced pain, all of which are attributes fordisease-modifying osteoarthritis drugs (DMOADs). Additionally, sometherapeutic agents have shown undesirable side effects at the dosagesrequired for therapeutic efficacy.

SUMMARY

Calcitonin products and therapies for treating inflammatory ordegenerative diseases in a subject are disclosed herein. Treatment maybe of a human or a non-human subject. In an embodiment, a therapy of thepresent disclosure permits a combination of therapeutic agents to beeffective at lower dosages than would be necessary if the therapeuticagents were used singly. In an embodiment, the use of lower dosagesresults in a lower risk of side effects that are sometimes associatedwith monotherapies.

According to aspects illustrated herein, there is provided a method fortreating a degenerative or an inflammatory disease that includesadministering calcitonin, in free or salt form, to a subject in need ofsuch treatment; and administering, as part of a combination therapy, asecond therapeutic agent for treatment of an inflammatory ordegenerative disease, the second therapeutic agent not being aglucocorticoid. In an embodiment, at least additive effectiveness isachieved by the combination relative to administering only the secondtherapeutic agent in the absence of the calcitonin.

According to aspects illustrated herein, there is provided a method fortreating a degenerative or an inflammatory disease in a subject thatincludes administering a therapeutically effective amount of calcitonin,in free or salt form, to the subject; and co-administering, as part of acombination therapy, a therapeutically effective amount of a COX-2inhibitor. In an embodiment, at least additive effectiveness is achievedby the combination relative to administering only the COX-2 inhibitor inthe absence of the calcitonin.

According to aspects illustrated herein, there is provided a method fortreating a degenerative or an inflammatory disease in a subject thatincludes administering a therapeutically effective amount of calcitonin,in free or salt form, to the subject; and co-administering, as part of acombination therapy, a therapeutically effective amount of a matrixmetalloproteinase inhibitor. In an embodiment, at least additiveeffectiveness is achieved by the combination relative to administeringonly the matrix metalloproteinase inhibitor in the absence of thecalcitonin.

According to aspects illustrated herein, there is provided a method fortreating a degenerative or an inflammatory disease in a subject thatincludes administering a therapeutically effective amount of calcitonin,in free or salt form, to the subject; and co-administering, as part of acombination therapy, a therapeutically effective amount of aninterleukin antagonist. In an embodiment, at least additiveeffectiveness is achieved by the combination relative to administeringonly the interleukin antagonists in the absence of the calcitonin.

According to aspects illustrated herein, there is provided a method fortreating a degenerative or an inflammatory disease in a subject thatincludes administering a therapeutically effective amount of calcitonin,in free or salt form, to the subject; and co-administering, as part of acombination therapy, a therapeutically effective amount of atetracycline antibiotic. In an embodiment, at least additiveeffectiveness is achieved by the combination relative to administeringonly the tetracycline antibiotic in the absence of the calcitonin.

According to aspects illustrated herein, there is provided a method fortreating a degenerative or an inflammatory disease that includesadministering a calcitonin, in free or salt form, to a subject in needof such treatment; and further administering, as part of a combinationtherapy, a second therapeutic agent selected from the group consistingof a protease inhibitor, an antibiotic, a non-steroidalanti-inflammatory agent, a COX-2 inhibitor and a steroidalanti-inflammatory agent other than a glucocorticoid.

According to aspects illustrated herein, there is provided a method forreducing the effective dosage (such as the approved dosage) of atherapeutic agent for treatment of an inflammatory or a degenerativedisease that includes administering the therapeutic agent andcalcitonin, in free or salt form, as part of a combination therapy, to asubject in need of treatment for the inflammatory or degenerativedisease.

According to aspects illustrated herein, there is provided a method fortreating an inflammatory or a degenerative disease that includesadministering to a subject in need of such treatment, as part of acombination therapy, (A) calcitonin, in free or salt form, and (B) asecond therapeutic agent for treatment of an inflammatory ordegenerative disease, wherein the second therapeutic agent isadministered at a dosage no higher than 90% of the normal dosage for thesecond therapeutic agent when administered alone.

According to aspects illustrated herein, there is provided a method fortreating a degenerative or an inflammatory disease that includesadministering calcitonin, in free or salt form, to a subject in need ofsuch treatment; and administering, as part of a combination therapy, anadditional agent selected from the group consisting of a proteaseinhibitor, an antibiotic, a non-steroidal anti-inflammatory agent, aCOX-2 inhibitor and a steroidal anti-inflammatory agent other thanglucocorticoid, wherein at least additive therapeutic effectiveness isachieved by the combination relative to administering only theadditional agent in the absence of calcitonin.

According to aspects illustrated herein, there is provided a method fortreating a degenerative or an inflammatory disease that includesadministering a therapeutically effective amount of calcitonin, in freeor salt form, to a subject in need of such treatment; and administering,as part of a combination therapy, a therapeutically effective amount ofa second therapeutic agent selected from the group consisting ofGalardin, Doxycycline and BB-94.

According to aspects illustrated herein, there is provided apharmaceutical composition that includes a first therapeutic agent thatis calcitonin, in free or salt form; a second therapeutic agent, whereinthe second therapeutic agent is not a glucocorticoid; and apharmaceutically acceptable excipient, carrier or diluent.

According to aspects illustrated herein, there is provided apharmaceutical composition that includes a first therapeutic agent thatis calcitonin, in free or salt form; a second therapeutic agent selectedfrom the group consisting of a protease inhibitor, an antibiotic, anon-steroidal anti-inflammatory agent, a COX-2 inhibitor and a steroidalanti-inflammatory agent other than glucocorticoid; and apharmaceutically acceptable excipient, carrier or diluent.

According to aspects illustrated herein, there is provided apharmaceutical composition that includes a first therapeutic agent thatis calcitonin, in free or salt form; a second therapeutic agent selectedfrom the group consisting of Galardin, Doxycycline and BB-94; and apharmaceutically acceptable excipient, carrier or diluent.

According to aspects illustrated herein, there is provided a kit for thetreatment of a degenerative or an inflammatory disease that includes afirst container having therein a first therapeutic agent that iscalcitonin, in free or salt form; and a second container having thereina second therapeutic agent, wherein the second therapeutic agent is nota glucocorticoid.

According to aspects illustrated herein, there is provided a kit for thetreatment of a degenerative or an inflammatory disease that includes afirst container having therein a first therapeutic agent that iscalcitonin, in free or salt form; and a second container having thereina second therapeutic agent selected from the group consisting of aprotease inhibitor, an antibiotic, a non-steroidal anti-inflammatoryagent, a COX-2 inhibitor and a steroidal anti-inflammatory agent otherthan glucocorticoid.

According to aspects illustrated herein, there is provided a kit for thetreatment of a degenerative or an inflammatory disease that includes aunit dosage of a first therapeutic agent that is calcitonin, in free orsalt form; and a unit dosage of a second therapeutic agent selected fromthe group consisting of a protease inhibitor, an antibiotic, anon-steroidal anti-inflammatory agent, a COX-2 inhibitor and a steroidalanti-inflammatory agent other than glucocorticoid.

According to aspects illustrated herein, there is provided a kit for thetreatment of a degenerative or an inflammatory disease that includes afirst container having therein a first therapeutic agent that iscalcitonin, in free or salt form; and a second container having thereina second therapeutic agent selected from the group consisting ofGalardin, Doxycycline and BB-94.

BRIEF DESCRIPTION OF THE DRAWINGS

The presently disclosed embodiments will be further explained withreference to the attached drawings.

FIG. 1 is a bar chart showing the effect of treatment on proteoglycanrelease from stimulated bovine articular cartilage explants, measured asGAGs. The normalized GAG release in the culture medium during the threeweek culture is shown. The dotted line represents the GAG release fromthe control condition to which all conditions are compared.

FIG. 2 is a bar chart showing the effect of treatment on collagenrelease from stimulated bovine articular cartilage explants, measured asCTX-II. The normalized CTX-II release in the culture medium during thethree week culture is shown. The dotted line represents the CTX-IIrelease from the control condition to which all conditions are compared.

FIG. 3 is a bar chart showing the effect of treatment on PGE2 releasefrom stimulated bovine articular cartilage explants. The normalized PGE2release in the culture medium during the three week culture is shown.The dotted line represents the PGE2 release from the control conditionto which all conditions are compared.

FIG. 4 is a bar chart comparing the effects of Galardin alone,calcitonin alone, and Galardin in combination with calcitonin onproteoglycan release in experiments involving bovine cartilage explants.Normalized data are shown.

FIG. 5 is a bar chart comparing the effects of Doxycycline alone,calcitonin alone, and Doxycycline in combination with calcitonin onproteoglycan release in experiments involving bovine cartilage explants.Normalized data are shown.

FIG. 6 is a bar chart comparing the effects of Doxycycline alone,calcitonin alone, and Doxycycline in combination with calcitonin oncollagen release in experiments involving bovine cartilage explants.Actual values are shown.

FIG. 7 is a bar chart comparing the effects of Doxycycline alone,calcitonin alone, and Doxycycline in combination with calcitonin onmatrix metalloproteinase (MMP) activity in experiments involving bovinecartilage explants. Actual values are shown.

FIG. 8 is a bar chart comparing the effects of Doxycycline alone,calcitonin alone, and Doxycycline in combination with calcitonin onprostaglandin E2 release in experiments involving bovine cartilageexplants. Normalized data are shown.

FIG. 9 is a bar chart comparing the effects of BB-94 alone, calcitoninalone, and BB-94 in combination with calcitonin on proteoglycan releasein experiments involving bovine cartilage explants. Normalized data areshown. Normalized data are shown.

FIG. 10 is a bar chart comparing the effects of BB-94 alone, calcitoninalone, and BB-94 in combination with calcitonin on collagen release inexperiments involving bovine cartilage explants. Actual values areshown.

FIG. 11 is a bar chart comparing the effects of BB-94 alone, calcitoninalone, and BB-94 in combination with calcitonin on matrixmetalloproteinase (MMP) activity in experiments involving bovinecartilage explants. Actual values are shown.

While the above-identified figures set forth presently disclosedembodiments, other embodiments are also contemplated, as noted in thediscussion. This disclosure presents illustrative embodiments by way ofrepresentation and not limitation. Numerous other modifications andembodiments can be devised by those skilled in the art which fall withinthe scope and spirit of the principles of the presently disclosedembodiments.

DETAILED DESCRIPTION

As used herein, the term “additive effect” and “additive effectiveness”refers to an effect in which two or more therapeutic active agents usedin combination produce a total effect essentially the same as the sum oftheir individual effects.

As used herein, the term “synergistic effect” refers to an effectarising between two or more therapeutic agents used in combination thatproduces an effect greater than the sum of their individual effects.

As used herein, the term “therapeutically effective amount” refers tothe amount/dose of an active therapeutic agent or a pharmaceuticaldosage form that is sufficient to produce an effective response (i.e., abiological or medical response of a tissue, system, animal or humansought by a researcher, veterinarian, medical doctor or other clinician)upon administration to a subject. The “therapeutically effective amount”will vary depending on inter alia the disease and its severity, and theage, weight, physical condition and responsiveness of the patient to betreated.

As used herein, “co-administering” and “co-administration” refers to theadministration of calcitonin and at least one additional therapeuticactive agent together. In some embodiments, one or more therapeuticactive agents may be formulated for oral administration (includingbuccal or sublingual) in solid or liquid form, for parenteral (includingsubcutaneous, intramuscular, intravenous, intraperitoneal,intra-arterial, or intradermal) route, for vaginal, nasal, topical(including buccal, sublingual or transdermal), or rectal administration.In some embodiments, one or more therapeutic active agents may beadministered orally utilizing oral dosage forms such as those disclosedin U.S. Pat. No. 6,086,918 (Stern et al), or as disclosed in pendingU.S. patent application Ser. No. 12/128,210 (Stern et al, published asUS 2009/0317462). In other embodiments, one or more therapeutic activeagents may be administered nasally using formulations such as those setforth in pending U.S. patent application Ser. No. 12/732,081 (Stern,published as US 2010/0256060). All of the therapeutic active agents usedin any of the therapies discussed herein may be formulated inpharmaceutical compositions which also include one or more of the othertherapeutic active agents. Alternatively, the therapeutic active agentsmay each be administered separately but sufficiently simultaneous intime so that a patient eventually has elevated blood levels or otherwiseenjoys the benefits of each of the active ingredients (or strategies)simultaneously. In an embodiment, one or more active ingredients are tobe formulated in a single pharmaceutical composition. In an embodiment,a kit is provided which includes at least two unit dosages, generallyprovided in separate sealed containers, wherein the contents of at leastone container differs, in whole or in part, from the contents of atleast one other container with respect to active ingredients containedtherein. Combination therapies discussed herein also include use of onetherapeutic active agent (of the combination) in the manufacture of amedicament for the treatment of the disease in question where thetreatment further includes another active ingredient of the combinationin accordance with the disclosure.

Except when otherwise noted or where apparent from context, dosagesherein refer to weight of active compounds unaffected by pharmaceuticalexcipients, diluents, carriers or other ingredients, although suchadditional ingredients are desirably included. Any dosage form (e.g.solid dosage forms: including powders, granules, tablets, capsules, andsuppositories; injections; nasal sprays; patches; depot injections orthe like) commonly used in the pharmaceutical industry may be used toadminister one or more active agents discussed herein, and the terms“excipient,” “diluent,” or “carrier” include such nonactive ingredientsas are typically included, together with active ingredients in suchdosage forms in the industry. For example, typical capsules, pills,enteric coatings, solid or liquid diluents or excipients, flavorants,preservatives, or the like may be included.

The present disclosure relates to combination therapies for thetreatment of an inflammatory or a degenerative disease, and particularlyto the use of calcitonin in combination with at least one other activeagent for treating such inflammatory or degenerative diseases, and totherapeutic products that include both calcitonin and at least one otheractive agent. The combination therapies of the present disclosure areused for the treatment of inflammatory or degenerative diseases such asinflammatory diseases of the joints and degenerative diseases of thejoints. Such diseases include, but are not limited to, osteoarthritis(OA), rheumatoid arthritis (RA), juvenile rheumatoid arthritis (JRA),lupus, ankylosing spondylitis (AS) and multiple sclerosis (MS). OA is adegenerative joint disease and a major cause of disability in theelderly. The hallmark of OA is the progressive destruction of articularcartilage (in addition to changes in subchondral bone, synovialmembrane, and other joint tissues). RA is an autoimmune disease thatinvolves pain and inflammation of the joints and surrounding tissues. Itcan also lead to loss of bone in the affected joints. Both OA and RA aremultimodal in nature.

Evidence suggests a direct anabolic effect of calcitonin on articularchondrocytes, resulting in increased proteoglycan synthesis. Theanticatabolic effects of calcitonin may involve induction of cAMP,resulting in attenuation of matrix metalloproteases (MMP)-mediatedcartilage degradation. This inhibition of metalloproteases has beensupported by measuring a decrease in C-terminal crosslinked telopeptidetype II collagen (CTX-II) levels which are a surrogate marker for TypeII collagen, a major constituent of articular cartilage.

In accordance with the present disclosure, calcitonin is used as part ofa combination therapy with at least one other therapeutic agenteffective to treat an inflammatory or a degenerative disease of thejoints. In an embodiment, such a combination therapy has additiveeffectiveness, relative to use of calcitonin alone or relative to use ofthe other therapeutic agent alone. In an embodiment, such a combinationtherapy has synergistic effectiveness, relative to use of calcitoninalone or relative to use of the other therapeutic agent alone. Thisevidence of additive or synergistic results relates to effectiveness onparameters expected to be markers of effectiveness in reducing processesimplicated in inflammatory or degenerative diseases. Because theseprocesses are multimodal, a combination therapy may be useful byproviding additive or synergistic effect on any one of the involvedprocesses.

In addition to enhanced effectiveness, in an embodiment a combinationtherapy of the present disclosure may, because of the additive orsynergistic effects, reduce the dosage required for therapeuticeffectiveness of the second (non-calcitonin) therapeutic agent, thusreducing the likelihood of side effects that have diminished thedesirability of these agents for some patients.

In an embodiment, a combination therapy of the present disclosureincludes an effective dose of calcitonin and an effective does of atleast one COX-2 inhibitor, where the effective dose of the COX-2inhibitor in the combination therapy is less than an effective dose of aCOX-2 inhibitor administered in a regular (i.e., non-combination)therapy protocol. Examples of COX-2 inhibitors include, but are notlimited to, valdecoxib, celecoxib, etoricoxib and rofecoxib.

In an embodiment, a combination therapy of the present disclosureincludes an effective dose of calcitonin and an effective does of atleast one MMP inhibitor, where the effective dose of the MMP inhibitorin the combination therapy is less than an effective dose of a MMPinhibitor administered in a regular (i.e., non-combination) therapyprotocol. Examples of MMP inhibitors include, but are not limited to,galardin, BB-94, and solimastat.

In an embodiment, a combination therapy of the present disclosureincludes an effective dose of calcitonin and an effective does of atleast one interleukin antagonist, where the effective dose of theinterleukin antagonist in the combination therapy is less than aneffective dose of an interleukin antagonist administered in a regular(i.e., non-combination) therapy protocol. Examples of an interleukinantagonist include anakinra (Kineret®).

In an embodiment, a combination therapy of the present disclosureincludes an effective dose of calcitonin and an effective does of atleast one antibiotic, where the effective dose of the antibiotic in thecombination therapy is less than an effective dose of an antibioticadministered in a regular (i.e., non-combination) therapy protocol.Examples of antibiotics include, but are not limited to, doxycycline,minocycline, oxytetracycline, enrofloxacin, ceftiofur, salinomycin, andtetracycline.

In an embodiment, a combination therapy of the present disclosureincludes an effective dose of calcitonin and an effective does of atleast one NSAID, where the effective dose of the antibiotic in thecombination therapy is less than an effective dose of an antibioticadministered in a regular (i.e., non-combination) therapy protocol.Examples of NSAID's include, but are not limited to, aspirin, ibuprofen,and naproxen

In some embodiments, the non-calcitonin second therapeutic agent used inthe combination therapy is used at a dosage no higher than 90 percent,no higher than 80 percent, no higher than 70 percent, of its recommendedor effective dosage when used alone.

All of the therapeutic agents used in any of the combination therapiesdisclosed herein may be formulated in pharmaceutical compositions whichalso include one or more of the other therapeutic agents. Alternatively,the therapeutic agents may each be administered separately butsufficiently simultaneous in time so that a subject eventually haselevated blood levels or otherwise enjoys the benefits of each of thetherapeutic agents simultaneously. In some embodiments of the presentdisclosure, for example, one or more therapeutic agents are to beformulated in a single pharmaceutical composition. Combination therapiesdiscussed herein also include use of one therapeutic agent of thecombination in the manufacture of a medicament for the treatment (orprevention) of the disease in question where the treatment or preventionfurther includes another therapeutic agents of the combination. While atleast two active therapeutic agents are required in the combinationtherapies of the present disclosure, more than two of the therapeuticagents discussed herein (e.g., three or more such agents) are alsocontemplated and are within the scope of the present disclosure.

In an embodiment of the present disclosure, a kit is provided whichincludes at least two separate containers, wherein the contents of atleast one container differs, in whole or in part, from the contents ofat least one other container with respect to active therapeutic agentscontained therein. Two or more different containers are used in thecombination therapies of the present disclosure. In an embodiment, thepresent disclosure relates to a kit for the treatment of a degenerativeor an inflammatory disease that includes a unit dosage form of a firsttherapeutic agent that is calcitonin, in free or salt form; and a unitdosage form of a second therapeutic agent, wherein the secondtherapeutic agent is not a glucocorticoid. Such a kit would normally(although not necessarily) include instructions for use. The kit mayinclude a plurality of the unit dosage forms.

The Calcitonin

The calcitonin of the present disclosure may be any natural calcitonin,or any truncate and/or modification thereof that retains calcitonin'santiresorptive properties. The calcitonins used according to the presentdisclosure also include variants, fragments and/or derivatives of acalcitonin peptide. The calcitonin may be in free or salt form.Calcitonin has been characterized from many animal sources. Many types,both natural and synthetic, are known, including but not limited tohuman calcitonin, salmon calcitonin, eel calcitonin or an analog thereof(elcatonin). Except where otherwise stated or apparent from context, theterm “calcitonin” as used herein is a generic term covering any and allcalcitonins, natural or synthetic, including the above-noted truncates,and regardless of whether in free or salt form.

In an embodiment, salmon calcitonin is used in accordance with thepresent disclosure for a number of reasons. Salmon Calcitonin (sCT) is anaturally occurring Amidated peptide hormone composed of 32 amino acidswhich binds to osteoclasts and inhibits bone resorption. Calcitoninsfrom many species are effective in humans, but salmon calcitonin is mostwidely used. Salmon calcitonin is highly potent in humans because of itshigh affinity for the human calcitonin receptor and its slow rate ofclearance. Thus, salmon calcitonin provides a number of advantages overeven human calcitonin, even when used as a pharmaceutical agent forhuman patients. Among the advantages provided by utilizing salmoncalcitonin instead of human calcitonin are increased potency, analgesiaand increased half-life. Also, lower dosages are necessary than withhuman calcitonin. There is substantial non-homology between salmon andhuman calcitonin, with only 50% identity in the amino acid sequences ofthe two calcitonins. Notwithstanding the foregoing preference for salmoncalcitonin, other calcitonins may be used in accordance with the presentdisclosure.

In an embodiment, when salmon calcitonin is chosen as the calcitonin fora combination therapy of the present disclosure and is delivered orallyas described above, concentration levels are between about 100micrograms (μg) to about 1000 μg of salmon calcitonin per dosage form.In an embodiment, when salmon calcitonin is chosen as the calcitonin fora combination therapy of the present disclosure and is delivered orallyas described above, concentration levels are between about 100 μg toabout 400 μg of salmon calcitonin per dosage form. In an embodiment,when salmon calcitonin is chosen as the calcitonin for a combinationtherapy of the present disclosure and is delivered orally as describedabove, concentration levels are between about 150 μg to about 300 μg ofsalmon calcitonin per dosage form.

The calcitonin may be delivered in any conventional manner commonlyutilized in the pharmaceutical industry. In an embodiment, thecalcitonin is administered nasally in accordance with the teachings ofStern, U.S. Pat. RE 40,812. Nasal calcitonin products are available,e.g. FORTICAL® (available from Upsher-Smith). Such nasal calcitoninformulations may be modified to add a second therapeutic agent of thecombination therapies discussed herein. In another embodiment, thecalcitonin is delivered orally in accordance with the teachings of Sternet al, U.S. Pat. No. 6,086,918; or pending U.S. patent application Ser.No. 12/128,210 (Stern et al, published as US 2009/0317462). Such oralcalcitonin formulations may be modified to add a second therapeuticagent of the combination therapies discussed herein. In someembodiments, the calcitonin and at least one additional therapeuticagent are combined in single pharmaceutical compositions. In someembodiments, the calcitonin and at least one additional therapeuticagent may be provided in unit dosage forms, and will generally beprovided in one or more sealed containers and may be provided as part ofa kit. Such a kit would normally (although not necessarily) includeinstructions for use. The kit may include a plurality of the unit dosageforms.

The Additional Agent of the Combination Therapy

The additional therapeutic agent of the present disclosure, which isused as part of a combination therapy with calcitonin, may be anytherapeutic agent that is individually effective in the treatment of oneor more inflammatory or degenerative diseases. These diseases includebut are not limited to osteoarthritis (OA), rheumatoid arthritis (RA),juvenile rheumatoid arthritis (JRA), lupus, ankylosing spondylitis (AS)and multiple sclerosis (MS). Classes of such agents include, but are notlimited to, antibiotics, steroidal anti-inflammatory agents,non-steroidal anti-inflammatory agents, COX-2 inhibitors, and proteaseinhibitors, especially MMP inhibitors that inhibit degradation ofType-II collagen and aggrecanase inhibitors. Examples of such agents foruse in a combination therapy of the present disclosure include, but arenot limited to, minocycline, doxycycline, oxytetracycline, enrofloxacin,ceftiofur, salinomycin, tetracycline, BB-94 (available from TocrisBioscience as BATIMASTAT®), solimastat, Galardin (available from U.S.Biological as LLOMASTAT®), anakinra (available from Biovitrum asKINERET®), valdecoxib, celecoxib (available from Pfizer as CELEBREX®),etoricoxib, rofecoxib, aspirin, ibuprofen and naproxen (available fromBayer as ALEVE®).

In an embodiment, the additional therapeutic agent is administered atdosages reported to be effective for the therapeutic agent whenadministered alone. With respect to commercial products, the therapeuticagent may be administered at dosages recommended by thelabeling/packaging of the product. However, it is believed thatcalcitonin may lower the effective dosage of the additional thetherapeutic agent. In some embodiments, the additional the therapeuticagent is administered at a dosage no higher than 90 percent of thenormal dosage, no higher than 80 percent of normal dosage, or in someembodiments, no higher than 70 percent of normal dosage. By reducingeffective dosage of the additional the therapeutic agent, undesirableside effects associated with long term use of such agents as, forexample, steroids, NSAIDs or COX-2 inhibitors, may be reduced.

The examples of combination therapies described in the followingExamples, which are set forth to aid in the understanding of thedisclosure, should not be construed to limit in any way the scope of thedisclosure as defined in the claims which follow thereafter. Thefollowing examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the described embodiments, and are not intended to limitthe scope of what the inventors regard as their invention nor are theyintended to represent that the experiments below are all or the onlyexperiments performed. Efforts have been made to ensure accuracy withrespect to numbers used (e.g. amounts, temperature, etc.) but someexperimental errors and deviations should be accounted for. Unlessindicated otherwise, parts are parts by weight, molecular weight isweight average molecular weight, temperature is in degrees Centigrade,and pressure is at or near atmospheric.

In an embodiment, the Examples below were carried out to identifywhether the combination of salmon calcitonin and other active agents canproduce additive or synergistic effects in reducing collagen degradationin a bovine in vitro collagen matrix model. Studies were designed toidentify synergistic effects of salmon Calcitonin (sCT) with referenceto treatments for osteoarthritis using the bovine cartilage explantmodel in which degradation is induced by a cocktail of three cytokines.Matrix degradation was quantified by measuring proteoglycan release,collagen release and MMP activity. The pro-inflammatory molecule PGE2was also measured. The studies consisted of two phases:

Phase 1: the highest ineffective dose of calcitonin and referencecompounds (Galardin, Kineret® (anakinra), Celebrex® (celecoxib),Dexamethasone, BB-94 and Doxycycline) were determined in a bovineexplant model for articular cartilage degradation.

Phase 2: the effect of calcitonin in combination with each of thereference compounds, in their highest ineffective dose, was studied todetermine their synergistic effects in blocking cartilage destruction.

The studies were designed to identify classes of compounds that worksynergistically to decrease parameters associated with the developmentand progression of OA and/or RA. These studies provide convincingdemonstration of significant trends for critical parameters known to beinvolved in the etiology and pathogenesis of OA and RA.

EXAMPLES Example 1

In this experiment, three or four concentrations of each compound weretested to find the highest ineffective dose, i.e., the concentration inwhich each compound has no or only a very small effect on cartilagedegradation.

Full thickness cartilage was obtained from the metacarpophalangealjoints of approximately 6 months old calves (from a localslaughterhouse). Cartilage punches (4 mm) were taken from the cartilageand placed in culture dishes with culture medium (DMEM+FCS, penicillin,streptomycin and ascorbic acid). Cartilage explants were weighed andexplants between about 5 and about 15 mg were divided into a 96-wellculture plate with 1 explant per well and 200 μL culture medium.Cartilage degradation was induced by addition of TNFα, IL-1α, and OSM at10 ng/mL each to the culture medium. Test agents were then added atdifferent concentrations. During three weeks of culture, the culturemedium and the test compounds were refreshed once weekly. In additionfresh calcitonin was added twice weekly to the cartilage explants.Culture medium and cartilage explants were stored at −20° C. untilanalysis. Each culture condition was cultured in four-fold and theentire experiment was performed on cartilage from three different paws.The different culture conditions are listed below in Table 1.

TABLE 1 Culture 10 ng/ml TNFa Condition 10 ng/ml IL-1a Compound Compoundnumber 10 ng/ml OSM name concentration 1 % % % 2 + — — 3 + Calcitonin 10nM 4 + Calcitonin 1 nM 5 + Calcitonin 0.3 nM 6 + Calcitonin 0.1 nM 7 +Galardin 10 μM 8 + Galardin 1 μM 9 + Galardin 0.1 μM 10 + Kineret ®(anakinra) 10 μg/ml 11 + Kineret ® (anakinra) 1 μg/ml 12 + Kineret ®(anakinra) 0.1 μg/ml 13 + Celebrex ® (celecoxib) 1 μM 14 + Celebrex ®(celecoxib) 0.1 μM 15 + Celebrex ® (celecoxib) 0.01 μM 16 +Dexamethasone 1 μM 17 + Dexamethasone 0.1 μM 18 + Dexamethasone 0.01 μM19 + Doxycycline 100 μM 20 + Doxycycline 10 μM 21 + Doxycycline 1 μM

Outcome parameters included (i) CTX-II in the culture medium as ameasure of collagen degradation, (ii) GAG in the culture medium andcartilage explant as a measure of proteoglycan degradation, (iii)prostaglandin E2 (PGE2) measured by ELISA in the culture medium as ameasure of inflammation, and (iv) general MMP activity in the culturemedium. Not all outcome parameters were determined for each of theculture conditions.

Collagen Release:

Collagen release was measured as CTX-II release per mg cartilage.Collagen was measured in the pooled medium using the CTXII ELISA assay(Pre-clinical Cartilaps®, Nordic Bioscience). Stimulation with thecombination of interleukin-1a (IL-1a), TNFα and oncostatin M (OSM)resulted in an upregulation of the CTX-II release from 14 to 135 ng/mgcartilage.

Proteoglycan Release:

The proteoglycan release is measured as GAG release per mg cartilage.Proteoglycan (GAG) content was determined in the pooled medium andexplants using the Blyscan® colorimetric assay (Biocolor, Belfast, UK).This value is normalized against the control condition of each donor tocorrect for donor variation. The basal release of unstimulated cartilageis 36% (p<0.05) when the stimulated (IL-1a, TNFα and OSM) control is setto 100%.

PGE2 Release:

The prostaglandin E2 release was measured as PGE2 release per mgcartilage. PGE2 was determined in the pooled medium using a PGE2 ELISAfrom R&D Systems. This value was normalized against the controlcondition of each donor to correct for donor variation. The basalrelease of unstimulated cartilage is 11% (p<0.01) when the stimulated(IL-1a, TNFα and OSM) control is set to 100%.

MMP Activity:

The MMP activity was measured as MMP activity per mg cartilage. GeneralMMP activity was determined in the pooled medium using the fluorogenicsubstrate TNO21 1-F. Stimulation with the combination of IL-1a, TNFα andOSM resulted in an upregulation of the MMP activity from 1 to 23 ng/mgcartilage.

Stimulation of cartilage degradation with cytokine cocktail increasedthe proteoglycan release from 20 to 92 μg GAG per mg cartilage, seenormalized data presented in FIG. 1. Cytokine stimulation increased thecollagen release from 12 to 155 ng CTX-II per mg cartilage, seenormalized data presented in FIG. 2. Cytokine stimulation increased thePGE2 release from 22 to 199 μg PGE2 per mg cartilage, see normalizeddata presented in FIG. 3. Each bar represents the mean of threeindividual paws with the standard deviation. The dotted line representsthe value from the control condition to which all conditions arecompared. Statistical analysis was performed on the normalized data,differences between groups were tested by ANOVA followed by LSD post-hoctest where # indicates p<0.05 and * indicates p<0.01.

Galardin inhibited collagen release and slightly inhibited proteoglycanrelease. Kineret® (anakinra) inhibited all outcome parameters. Celebrex®(celecoxib) inhibited PGE2 release and had a small reducing effect onproteoglycan and collagen release. Dexamethasone inhibited all outcomeparameters. Doxycycline inhibited collagen and PGE2 release and had aslight reducing effect on proteoglycan release. The highest ineffectiveconcentration can differ depending on the outcome parameter. Based onthe results of phase I, the highest ineffective concentration wasdetermined as illustrated in Table 2. Table 2 provides, for anembodiment of the present disclosure, suggested concentrations for eachcompound.

TABLE 2 Proteoglycan PGE2 Suggested Compound Name release CollagenRelease release Concentrations Calcitonin >10 nM 0.3-1 nM >10 nM 0.5 nMGalardin 1-10 μM <0.1 μM >10 μM 0.05 μM Kineret ® (anakinra) <0.1 μg/ml<0/1 μg/ml <0.1 μg/ml 0.01 μM Celebrex ® (celecoxib) 0.1-1 μM >10 μM<0.01 μM 0.05 μM. Dexamethasone <0.01 μM <0.01 μM <0.01 μM 1 nMDoxycycline >100 μM 10-100 μM 1-10 μM 10 μM

Example 2

In this experiment the synergistic effects of Calcitonin in combinationwith other active agents on cartilage degradation were determined. Theresults from Example 1 were used to select the concentrations of thecompounds for this Example.

Full thickness cartilage was obtained from the metacarpophalangealjoints of approximately 6 months old calves (from a localslaughterhouse). Cartilage punches (4 mm) were taken from the cartilageand placed in culture dishes with culture medium (DMEM+FCS, penicillin,streptomycin and ascorbic acid). Cartilage explants were weighed andexplants between about 5 and about 15 mg were divided into a 96-wellculture plate with 1 explant per well and 200 μL culture medium.Cartilage degradation was induced by addition of TNFα, IL-1a, and OSM at10 ng/mL each to the culture medium to induce cartilage degradation.Test agents were then added at different concentrations. During threeweeks of culture, the culture medium and the test compounds wererefreshed once weekly. In addition fresh calcitonin was added twiceweekly to the cartilage explants. Culture medium and cartilage explantswere stored at −20° C. until analysis. Each culture condition wascultured in four-fold and the entire experiment was performed oncartilage from three different paws. The different culture conditionsare listed below in Table 3.

TABLE 3 10 ng/ml TNFa Condition 10 ng/ml IL-1a Compound 1 Compound 2number 10 ng/ml OSM Name Concentration Name Concentration 1 − — — — —2 + — — — — 3 + Calcitonin 0.5 nM — — 4 + 10 nM — — 5 + Galardin 50 nM —— 6 + 10 μM — — 7 + Celebrex ® 0.1 μM — — 8 + (celecoxib) 1 μM — — 9 +Dexamethasone 1 nM — — 10 + 5 μM — — 11 + Doxycycline 10 μM — — 12 + 100μM — — 13 + BB-94 10 nM — — 14 + 100 nM — — 15 + Calcitonin 0.5 nMGalardin 50 nM 16 + 10 nM 10 μM 17 + Calcitonin 0.5 nM Celebrex ® 0.1 μM18 + 0.5 nM (celecoxib) 1 μM 19 + 10 nM 0.1 μM 20 + 10 nM 1 μM 21 +Calcitonin 10 nM Dexamethasone 1 nM 22 + 10 nM 5 μM 23 + Calcitonin 0.5nM Doxycycline 10 μM 24 + 10 nM 10 μM 25 + 10 nM 100 μM 26 + Calcitonin0.5 nM BB-94 10 nM 27 + 0.5 nM 100 nM 28 + 10 nM 10 nM 29 + 10 nM 100 nM

FIG. 4 illustrates the effect of Calcitonin and Galardin, individuallyand in combination, on proteoglycan release. Each bar represents themean of three individual paws with the standard deviation. Normalizeddata is shown. Differences between groups were tested by ANOVA followedby LSD post-hoc test where “#” indicates p<0.05 and “*” indicatesp<0.01.

As shown in FIG. 4, 10 nM Calcitonin had an effect on the normalized GAGrelease (reduced the outcome parameter from 100% to 87%), whereas 10 μMof Galardin reduced the outcome parameter significantly (p<0.01) from100% to 72%. The combination of Calcitonin and Galardin resulted in asignificant (p<0.01) reduction to 65%.

FIGS. 5-8 show the effect of Calcitonin and Doxycycline, individuallyand in combination, on proteoglycan release (FIG. 5), collagen release(FIG. 6), MMP activity (FIG. 7) and PEG release (FIG. 8). Each barrepresents the mean of three individual paws with the standarddeviation. For the proteoglycan release the normalized data is shown,while for collagen release and MMP activity the actual values are shownbecause normalization was not possible. Differences between groups weretested by ANOVA followed by LSD post-hoc test where “#” indicates p<0.05and “*” indicates p<0.01.

As shown in FIG. 5, 10 nM Calcitonin slightly decreased the GAG releasefrom 100% to 87% and 100 μM Doxycycline decreased the outcome parameterto 80%. The combination of Calcitonin and Doxycycline resulted a furtherreduction to 73%.

As shown in FIG. 6, 0.5 nM Calcitonin slightly decreased the CTX-IIrelease from 135 to 133 ng/mg cartilage and 10 μM Doxycycline decreasedthe outcome parameter to 125 ng/mg cartilage. The combination ofCalcitonin and Doxycycline resulted a further reduction to 73 ng/mgcartilage, suggesting a synergistic effect. This effect is stronger thanexpected based on the effects of the compounds alone.

As shown in FIG. 7, 10 nM Calcitonin decreased the MMP activity from 23to 15 RFU/sec per mg cartilage and 10 μM Doxycycline decreased theoutcome parameter to 14 RFU/sec per mg cartilage. The combination ofCalcitonin and Doxycycline resulted a further reduction to 7 RFU/sec permg cartilage.

As shown in FIG. 8, 10 nM Calcitonin increased the PGE2 release from 100to 117% and 10 μM Doxycycline to 111%. The combination of Calcitonin andDoxycycline resulted a further increase to 145%.

The combination of Calcitonin and Doxycycline seemed to have a largereffect on all outcome parameters than the compounds alone. The strongesteffect of this combination was observed on collagen release. Thecombination of Calcitonin and Doxycycline tended towards a decrease inMMP activity, proteoglycan and collagen release more then Doxycyclinealone. The PGE2 release was further increased by the combination ofCalcitonin and Doxycycline.

FIGS. 9-11 show the effect of Calcitonin and BB-94, individually and incombination, on proteoglycan release (FIG. 9), collagen release (FIG.10) and MMP activity (FIG. 11). Each bar represents the mean of threeindividual paws with the standard deviation. For the proteoglycanrelease the normalized data is shown, while for collagen release theactual values are shown because normalization was not possible.Differences between groups were tested by ANOVA followed by LSD post-hoctest where “#” indicates p<0.05 and “*” indicates p<0.01.

As shown in FIG. 9, 10 nM Calcitonin reduced the normalized proteoglycanrelease from 100 to 87%. BB-94 lowered the proteoglycan release to 83and 80% at 10 nM and 100 nM respectively. Combining the two compoundsresulted in a reduction to 97 and 90% at 10 nM and 100 nM BB-94respectively.

As shown in FIG. 10, 0.5 nM Calcitonin decreased the CTX-II release from135 to 133 ng/mg cartilage. BB-94 changed CTX-II release to 137 and 27ng/mg cartilage at 10 nM and 100 nM respectively. Combining the twocompounds resulted in a reduction to 88 and 11 ng/mg cartilage at 10 nMand 100 nM BB-94 respectively. This effect is stronger than expectedbased on the effects of the compounds alone.

As shown in FIG. 11, 10 nM Calcitonin decreased the MMP activity from 23to 15 RFU/sec per mg cartilage. BB-94 lowered the MMP activity to 13 and4 RFU/sec per mg cartilage at 10 nM and 100 nM respectively. Combiningthe two compounds resulted in a reduction to 11 and 3 RFU/sec per mgcartilage 10 nM and 100 nM BB-94 respectively.

All patents, patent applications, and published references cited hereinare hereby incorporated by reference in their entirety. It will beappreciated that various of the above-disclosed and other features andfunctions, or alternatives thereof, may be desirably combined into manyother different systems or applications. Various presently unforeseen orunanticipated alternatives, modifications, variations, or improvementstherein may be subsequently made by those skilled in the art which arealso intended to be encompassed by the following claims.

What is claimed is:
 1. A method for treating a degenerative or aninflammatory disease in a subject comprising: administering atherapeutically effective amount of calcitonin, in free or salt form, tothe subject; and co-administering, as part of a combination therapy, atherapeutically effective amount of a second therapeutic agent selectedfrom the group consisting of a matrix metalloproteinase inhibitor, aninterleukin antagonist, an aggrecanase inhibitor, a tetracylineantibiotic, and a non-steroidal anti-inflammatory drug (NSAID) to thesubject.
 2. The method of claim 1 wherein the calcitonin is selectedfrom the group consisting of human calcitonin, salmon calcitonin, eelcalcitonin and elcatonin.
 3. The method of claim 1 wherein thecalcitonin is salmon calcitonin.
 4. The method of claim 1 wherein thematrix metalloproteinase inhibitor is selected from one of galardin orBB-94.
 5. The method of claim 1 wherein the calcitonin and the secondtherapeutic agent are each administered to the subject orally.
 6. Themethod of claim 1, wherein the NSAID is selected from the groupconsisting of aspirin, ibuprofen, naproxen, valdecoxib, celecoxib,etoricoxib and rofecoxib.
 7. The method of claim 6 wherein thecalcitonin is selected from the group consisting of human calcitonin,salmon calcitonin, eel calcitonin and elcatonin.
 8. The method of claim6 wherein the calcitonin is salmon calcitonin.
 9. The method of claim 1wherein the tetracycline antibiotic is doxycycline.
 10. The method ofclaim 9 wherein the calcitonin and the tetracycline antibiotic are eachadministered to the subject orally.
 11. A pharmaceutical composition forthe treatment of a degenerative or an inflammatory disease comprising: afirst therapeutic agent that is calcitonin, in free or salt form; asecond therapeutic agent selected from the group consisting of a matrixmetalloproteases inhibitor, an interleukin antagonist, an aggrecanaseinhibitor, a tetracyline antibiotic, and a non-steroidalanti-inflammatory drug (NSAID); and a pharmaceutically acceptableexcipient, carrier or diluent.
 12. The pharmaceutical composition ofclaim 11 wherein the second therapeutic agent is a matrixmetalloproteases inhibitor.
 13. The pharmaceutical composition of claim11 wherein the second therapeutic agent is an interleukin antagonist.14. The pharmaceutical composition of claim 11 wherein the secondtherapeutic agent is an aggrecanase inhibitor.
 15. The pharmaceuticalcomposition of claim 11 wherein the second therapeutic agent is atetracyline antibiotic.
 16. The pharmaceutical composition of claim 11wherein the second therapeutic agent is a NSAID.
 17. The pharmaceuticalcomposition of claim 11 wherein the second therapeutic agent isadministered at a dosage no higher than 90% of the normal dosage for thesecond therapeutic agent when administered alone.
 18. The pharmaceuticalcomposition of claim 11, wherein the composition is an aqueous nasalspray.
 19. The pharmaceutical composition of claim 11, wherein thecomposition is a solid dosage form.
 20. The pharmaceutical compositionof claim 11 wherein the calcitonin is salmon calcitonin.
 21. The methodof claim 1, wherein the calcitonin and the second therapeutic agent areadministered as a single pharmaceutical composition.
 22. The method ofclaim 1, wherein the calcitonin and the second therapeutic agent areadministered separately.
 23. The method of claim 1, wherein the secondtherapeutic agent is administered at a dosage no higher than 90% of thenormal dosage for the second therapeutic agent when administered alone.